The physical characterization of the iron site in low-spin ferric heme proteins is very important in working toward an understanding of the structure-function relationships in these systems. The proposed research will investigate two aspects of the protein structure: i) how the ligand field of the iron is perturbed by slight changes in the amino acid sequence and structure of the protein, and ii) the temperature dependence of the spin-lattice relaxation and how it varies with changes in the solvent. The first line of investigation is made possible by the expression of a synthetic rat hepatic cytochrome b5 gene in E. coli, where it is expressed in extremely large quantities, up to 20% of total cell protein. Specific modifications can be made in the cytochrome by cartridge mutagenesis. These modified proteins will be studied by EPR and Mossbauer spectroscopy, and the spectra will be parametrized by a S=1/2 Spin Hamiltonian, which can then be used as the basis for a ligand field characterization of the iron site. The temperature dependence of the Mossbauer spectra will serve as a further test of the fractal model for protein vibrational modes. EPR measurements of various heme and iron-sulfur proteins indicate that the phonon density of states in these systems is consistent with a model in which the protein has a fractional dimension of 1.1-1.8. These measurements were made for temperatures below 12K. Mossbauer spectroscopy is an ideal method to extend these studies to the temperature range 15K- 200K because 1) the integrated intensity of the Mossbauer spectrum is not as strong a function of temperature as is EPR; and, 2) EPR measurements indicate that the fluctuation rates for these temperatures should be in the range to which Mossbauer spectra are sensitive. We propose to first study myoglobin azide and the effects of the solvent on its fractional dimension, with the work later to be extended to the Horseradish peroxidase cyanide complex, cytochrome b5, and small heme model compounds.